Despite the encouraging results of non-living vaccines in experimental models, including defined DNA-based and recombinant protein-based vaccines, a safe, non-living vaccine has consistently failed to confer significant protection against cutaneous or visceral leishmaniasis in human trials. While there are apt to be many explanations for these discrepant outcomes, one key difference between the laboratory and field-based trials is the use of needle challenge to evaluate vaccines in animals, as compared to natural sand fly challenge in the human trials. Remarkably, no experimental vaccine has been evaluated by sand fly challenge in a controlled, laboratory setting. In addition to the delivery of infectious stage parasites into the dermis, infected sand flies also deposit pharmacologically active saliva, and parasite-derived secreted glycoproteins, each known to enhance the severity of disease when co-administered with parasites by needle. Furthermore, we have recently reported that sand fly transmission induces a qualitatively unique inflammatory response at the bite site that includes a dynamic recruitment of neutrophils and that these neutrophils markedly enhance the ability of parasites to establish primary infection. We have produced a clear set of data to indicate that the non-living vaccines that confer powerful protection against needle challenge are ineffective against sand fly challenge. The difference is not accounted for by the quality or dose of infectious stage parasites in the respective inocula. Neutrophil depletion following sand fly challenge rescued the ability of the killed vaccine to confer protection. Analysis at 2 weeks post-transmission revealed that neutrophil depletion resulted in increased Ag-specific interferon gamma (IFNg) production by CD4+ T cells. These findings demonstrate that the early recruitment and persistence of high numbers of neutrophils at the bite site seriously compromise the expression of secondary immunity in the skin. [unreadable] [unreadable] As neutrophils are the first cell type to take up Leishmania following transmission by bite, and as their specific depletion enhances host resistance to L. major, the possibility that infected neutrophils can encounter immature dendritic cells (iDCs) and compromise their function has been explored. Following i.d. inoculation of RFP-expressing L. major into lysozyme-GFP mice, the recovered GFP+ neutrophils were sorted into uninfected and infected populations, and co-cultured overnight with iDC. Following an additional sort of GFP+ and GFP- iDC, we investigated the priming capacity of these different populations of DC by co-culture with OT-I CD8+ T cells in the presence of soluble OVA. Uptake of infected neutrophils inhibited DC maturation, and completely eliminated their capacity to stimulate OT-I cells to proliferate and release IFNg. Their dysfunction was far more pronounced than the DC that had taken up uninfected neutrophils, a difference that correlated with the enhanced apoptotic status of the infected neutrophils, as determined by annexin staining. In vivo, neutrophil depletion increased the proliferation of adoptively transferred OT-I cells in response to infection by L. major transgenic parasites expressing OVA. These data demonstrate both a novel mechanism by which Leishmania parasites can induce immune dysregulation and a previously unrecognized role of infected neutrophils during the immune response to L. major. [unreadable] [unreadable] Endogenous IL-10 is a central mediator of immune homeostasis, necessary to keep in check the strong inflammatory reactions that can accompany the expression of anti-microbial immunity in local tissues. A consequence of the balance struck between host immunity and pathology can be chronic or persistent infection, and the absence of IL-10 has been shown to result in more efficient clearance of a variety of pathogens, including L. major. We recently reported on the inability of conventionally resistant C57BL/6 mice to successfully resolve infection by an isolate of L. major, despite a strong IFNg response by the host. Susceptibility was caused by antigen-specific IL-10 from CD4+ cells that were also producing IFNg. In followup studies, we have explored the role for IL-27 in the regulation of IL-10 from Th1 cells in Leishmaniasis. IL-27 was found to enhance IL-10 induction in L. major primed, committed Th1 cells in vitro. Cytokine analysis of CD4+ cells in the lesions and draining lymph nodes of infected IL-27R deficient (WSX-1-/-) mice revealed diminished IL-10 from IFNg+ CD4+ cells, which was accompanied by a reduction in total IFNg+ CD4+ cells and an increase in IL-4. Despite the inhibition of IL-10 from CD4+ cells, no significant change in parasite numbers was observed, due both to the dysregulated IL-4 and residual levels of IL-10. Strikingly, infected WSX-1-/- mice developed severe lesions that were associated with the appearance of IL-17+ CD4+ cells, demonstrating a function for IL-27 in blocking the development of inappropriate Th17 cells during L. major infection. The results demonstrate the pleiotropic effects that IL-27 has on L. major-driven Th1, Th2, and Th17 development, and reinforce its function as a key regulatory cytokine that controls the balance between immunity and pathology. [unreadable] [unreadable] The mechanisms underlying the failure to control the growth and systemic spread of Leishmania parasites in human visceral leishmaniasis (VL) are not well understood. Produced as a possible homeostatic mechanism to control persistent infection-induced inflammation, elevated levels of the suppressive cytokine IL-10 have repeatedly been observed in the spleen, bone marrow, PBMC, and plasma of VL patients with active disease. Within the parasitized spleen, the source ofthe elevated IL-10 mRNA was shown to be CD3+CD25-Foxp3- cells. Direct demonstration that IL-10 impairs antigen-specific IFNg responses and promotes parasite growth in a target organ such as the spleen would have strong relevance to the pathogenesis of VL. We have taken advantage of the presence of infected macrophages within the biopsied splenic cells as a readout for IL-10 function. The small volume of residual splenic aspirate obtained for diagnosis was split into two equal volumes, and cultured in the presence of anti-IL-10 antibodies or isotype control Ig. After 3 days, the number of viable parasites was quantified by serial dilution onto blood agar plates. Within the 18 paired samples for which parasites could be grown out, 14 had fewer parasites following the 3 day incubation with anti-IL-10, and in 6 of the samples, the presence of the anti-IL-10 antibodies promoted complete killing of the parasite. Multiplex analysis of cytokines present within the culture supernatants of the splenic cells revealed a significant increase in both TNF-alpha and IFNg levels in the anti-IL-10 treated cultures, suggesting IL-10 suppression of T cell effector function as one mechanism to account for compromised immunity in these patients. These clinical findings, along with those in experimental models showing that anti-IL-10 receptor antibody promotes rapid control of L. donovani infection and dramatically enhances the leishmanicidal activity of pentavalent antimony, clearly demonstrate the potential benefits of IL-10 inhibition as an approach to therapy in VL.